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Cloning-free CRISPR protocols for C. elegans

INTRODUCTION:

A major goal of genetic engineering is to develop methods to modify the genome of animals easily and efficiently. CRISPR/Cas9 technology has greatly accelerated progress in this field by providing a simple method to introduce double-strand breaks in the genome. If a DNA with homology to the break (homology arms) is introduced at the same time as Cas9, the cell’s DNA repair machinery will use that DNA to repair the break and copy the sequence between the homology arms into the genome (homology-dependent repair - HDR).Our lab has found that, in C. elegans, homology-dependent repair is remarkably efficient (~100% of breaks repaired by HDR) provided that the repair templates are LINEAR and have short (~35 bases) homology arms that directly flank the Cas9 cleavage site (Paix et al., 2014, 2015).Linear DNAs (ssODNs and PCR amplicons) with 35-base homology arms are sufficient to introduce base- and gene-sized edits (up to 1.6kb).On the basis of these findings, we have developed a cloning-free protocol for editing the C. elegans genome. Our protocol does not require selection markers and uses the same experimental approach to mutate, tag, or delete any gene in the genome.

PROTOCOL OVERVIEW:

Requirements:

A Cas9 recognition site(s) within 20 bases of the edit.

An edit that will fit into a template <2kb long.

Experimental outline:

Day 0: Order gene-specific reagents:

- crRNA(s) for Cas9 (can also be made in house) - ssODNs to use directly as template or to amplify template

You will also need Cas9 protein and tracr RNA. Both are available commercially or can be made in house. See below for protocols to make your own Cas9 protein and crRNA.